脂联素在L-02细胞中对肝糖代谢信号转导机制的研究
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摘要
目的：在人肝细胞株L-02中获取具有生物学活性的脂联素重组蛋白的分泌性表达,并在L-02细胞中探讨脂联素抑制糖异生的信号转导机制。
方法：①从人脂肪组织中提取总RNA,利用RT-PCR方法得到人脂联素基因cDNA序列,自行设计引物一对,经PCR扩增人脂联素基因完全编码序列,构建pMD18-T/ADPN重组克隆质粒,目的序列测序鉴定；PCR扩增脂联素目的片段,连至真核表达载体pCDNA3.1/CT-GFP-TOPO,构建脂联素真核表达质粒pCDNA3.1/CT-GFP-TOPO-ADPN。②将pCDNA3.1/CT-GFP-TOPO-ADPN转化入TOP10感受态细胞,筛选可能含有阳性克隆的白色菌落进行鉴定,提取阳性克隆中的重组质粒,命名为pCDNA3.1-ADPN。③将pCDNA3.1—ADPN经脂质体介导、G418筛选后转染至人肝细胞株L-02,免疫荧光法观察转染率,收获的细胞裂解液及细胞培养上清经SDS-PAGE、western blot、细胞免疫组织化学法检测脂联素表达情况。④用含0 mmol/L、5 mmol/L、15 mmol/L和30 mmol/L葡萄糖的培基分别模拟无糖、低糖、中糖、高糖环境。将转染pCDNA3.1—ADPN的L-02细胞和对照组分别在上述环境中处理24 h,酶学法测定培养基血糖,观察重组脂联素蛋白的活性。⑤以western blot测定正常L-02组、转染组、30 mmol/L glucose处理组和“饥饿”组(72 h未换液)中phospho-AMPK、及其下游环腺苷酸反应元件结合蛋白(CREB)、phospho-CREB蛋白表达情况,免疫组化法观察四组细胞中环腺苷酸反应元件结合蛋白辅激活物2(TORC2)的定位情况,葡萄糖脱氢酶偶联法测定葡萄糖-6-磷酸酶(G6P)活性。
结果：①成功构建脂联素克隆质粒pMD18-T/ADPN和真核表达质粒pCDNA3.1/CT-GFP-TOPO-ADPN(简称pCDNA3.1-ADPN)。②转染pCDNA3.1-ADPN的L-02细胞在免疫荧光显微镜下可见绿色荧光,表明转染成功,转染率在60%以上。③在细胞培养上清和沉淀中均检测到表达产物,表明重组蛋白利用自身信号肽获得分泌性表达。细胞裂解液和细胞培养上清中表达的重组蛋白分子量约为57 kD,具有人脂联素抗原性。④转染pCDNA3.1—ADPN的L-02细胞高糖(30mmol/L)处理24 h后其培基血糖低于对照组(p<0.05),但在无糖、低糖和中糖环境中无明显差异(p>0.05)。⑤与其它组比较,转染组：phospho-AMPK蛋白表达升高(p<0.05), TORC2主要在定位在细胞质中,G6P酶的活性显著降低(p<0.05),但CREB、phospho-CREB蛋白表达无明显差异(p>0.05)。
结论：我们成功构建了人脂联素真核表达质粒,并在人肝细胞株L-02中得到可溶性、分泌性表达,获得的重组蛋白具有生物学活性。脂联素抑制糖异生的机制可能与活化phospho-AMPK,抑制TORC2进入核内,从而抑制糖异生酶G6P的表达有关,其信号转导通路可能为Adiponectin-AMPK-CREB-TORC2-糖异生关键酶。这为进一步研究脂联素的信号通路,并探寻新的信号分子奠定了坚实的基础。
Objectives:To obtain the expression of recombinant adiponection protein which has biologic activity in human hepatocyte line named L-02. And to study the signaling pathway of adiponectin modulates gluconeogenesis in human hepatocyte L-02.
Methods:①To extract the total RNA from human adipose tissue. We got the adiponectin cDNA by RT-PCR. Design a pair of primers to amplify the encoding fragment of human ADPN gene by PCR. Construct recombinant clone vector pMD18-T/ADPN, and identify the sequence. Extract the encoding fragment of human ADPN gene, ligated into eukaryotic expression vector pCDNA3.1/CT-GFP-TOPO, construct adiponectin eukaryotic expression vector pCDNA3.1/CT-GFP-TOPO-ADPN.②After transformation, recombinant expression vector pCDNA3.1/CT-GFP-TOPO-ADPN is introduced into TOP10 Competent cells. Choose the white colony to verify. Extract adiponectin recombination vector named pCDNA3.1-ADPN.③The cultured L-02 cells are transfected with pCDNA3.1-ADPN by liposome, after sieving by G418. Immunofluorescence observes transfection efficiency. The expression product in cell lysate and culture supernatant was identified by SDS-PAGE、western blot and immunohistochemical method.④Using 0mmol/L、5 mmol/L、15mmol/L and 30mmol/L glucose to simulate no glucose,low glucose, middle glucose, high glucose environment. Treat transfect ion cells and control cells with the above-mentioned conditions. Determine cultivate glucose by enzymology; detect the recombination adiponectin protein's activity.⑤Determine phospho-AMPK, CREB, phospho-CREB in normal L-02 group, transfected group, high glucose treatment group, and hungry group by western blot, observed the TORC2 localization in the four group, glucose dehydrogenase couple way detectes Glucose-6-phosphorate (G6P) activity.
Results:①Successfully construct the adiponectin clone vector pMD18-T/ADPN and adiponectin eukaryotic expression vector pCDNA3.1/CT-GFP-TOPO-ADPN. ADPN gene was inserted in correct location.②We can see the green fluorescence in the transfected pCDNA3.1/CT-GFP- TOPO-AND cells by the immunofluorescence microscope; the transfection efficiency is about 60%.③Recombinant protein secretes into culture medium. The MW of recombinant protein is about 57kD. Western blot proves that expressed protein could combine with ADPN polyclonal antibody.④There is obvious glucose difference between transfection group and control group (p<0.05), which treated with high glucose for 24 h. There is no obvious among no glucose group, low glucose group and middle glucose group (p>0.05).⑤Compare with other groups, there is stronger p-AMPK expression in the transfect ion group (p<0.05), TORC2 was mainly localization in cytoplasm, the G6P activity was cut down significant (p<0.05), but there is no significant in the expression of CREB、p-CREB (p>0.05).
Conclusions:Adiponectin eukaryotic expression vector was successfully constructed. It can express solubility and secrete adiponectin protein in human hepatocytes L-02. The recombinant protein has biology activity.The mechanism of inhibitin hepar glyconeogenesis may involve activing AMPK, depressing the TORC2 entry the nuclear, and cutting down the G6P activity. The signaling pathway may be adiponectin-AMPK-CREB-TORC2- glyconeogenesis key enzyme.
方法：①从人脂肪组织中提取总RNA,利用RT-PCR方法得到人脂联素基因cDNA序列,自行设计引物一对,经PCR扩增人脂联素基因完全编码序列,构建pMD18-T/ADPN重组克隆质粒,目的序列测序鉴定；PCR扩增脂联素目的片段,连至真核表达载体pCDNA3.1/CT-GFP-TOPO,构建脂联素真核表达质粒pCDNA3.1/CT-GFP-TOPO-ADPN。②将pCDNA3.1/CT-GFP-TOPO-ADPN转化入TOP10感受态细胞,筛选可能含有阳性克隆的白色菌落进行鉴定,提取阳性克隆中的重组质粒,命名为pCDNA3.1-ADPN。③将pCDNA3.1—ADPN经脂质体介导、G418筛选后转染至人肝细胞株L-02,免疫荧光法观察转染率,收获的细胞裂解液及细胞培养上清经SDS-PAGE、western blot、细胞免疫组织化学法检测脂联素表达情况。④用含0 mmol/L、5 mmol/L、15 mmol/L和30 mmol/L葡萄糖的培基分别模拟无糖、低糖、中糖、高糖环境。将转染pCDNA3.1—ADPN的L-02细胞和对照组分别在上述环境中处理24 h,酶学法测定培养基血糖,观察重组脂联素蛋白的活性。⑤以western blot测定正常L-02组、转染组、30 mmol/L glucose处理组和“饥饿”组(72 h未换液)中phospho-AMPK、及其下游环腺苷酸反应元件结合蛋白(CREB)、phospho-CREB蛋白表达情况,免疫组化法观察四组细胞中环腺苷酸反应元件结合蛋白辅激活物2(TORC2)的定位情况,葡萄糖脱氢酶偶联法测定葡萄糖-6-磷酸酶(G6P)活性。
结果：①成功构建脂联素克隆质粒pMD18-T/ADPN和真核表达质粒pCDNA3.1/CT-GFP-TOPO-ADPN(简称pCDNA3.1-ADPN)。②转染pCDNA3.1-ADPN的L-02细胞在免疫荧光显微镜下可见绿色荧光,表明转染成功,转染率在60%以上。③在细胞培养上清和沉淀中均检测到表达产物,表明重组蛋白利用自身信号肽获得分泌性表达。细胞裂解液和细胞培养上清中表达的重组蛋白分子量约为57 kD,具有人脂联素抗原性。④转染pCDNA3.1—ADPN的L-02细胞高糖(30mmol/L)处理24 h后其培基血糖低于对照组(p<0.05),但在无糖、低糖和中糖环境中无明显差异(p>0.05)。⑤与其它组比较,转染组：phospho-AMPK蛋白表达升高(p<0.05), TORC2主要在定位在细胞质中,G6P酶的活性显著降低(p<0.05),但CREB、phospho-CREB蛋白表达无明显差异(p>0.05)。
结论：我们成功构建了人脂联素真核表达质粒,并在人肝细胞株L-02中得到可溶性、分泌性表达,获得的重组蛋白具有生物学活性。脂联素抑制糖异生的机制可能与活化phospho-AMPK,抑制TORC2进入核内,从而抑制糖异生酶G6P的表达有关,其信号转导通路可能为Adiponectin-AMPK-CREB-TORC2-糖异生关键酶。这为进一步研究脂联素的信号通路,并探寻新的信号分子奠定了坚实的基础。
Objectives:To obtain the expression of recombinant adiponection protein which has biologic activity in human hepatocyte line named L-02. And to study the signaling pathway of adiponectin modulates gluconeogenesis in human hepatocyte L-02.
Methods:①To extract the total RNA from human adipose tissue. We got the adiponectin cDNA by RT-PCR. Design a pair of primers to amplify the encoding fragment of human ADPN gene by PCR. Construct recombinant clone vector pMD18-T/ADPN, and identify the sequence. Extract the encoding fragment of human ADPN gene, ligated into eukaryotic expression vector pCDNA3.1/CT-GFP-TOPO, construct adiponectin eukaryotic expression vector pCDNA3.1/CT-GFP-TOPO-ADPN.②After transformation, recombinant expression vector pCDNA3.1/CT-GFP-TOPO-ADPN is introduced into TOP10 Competent cells. Choose the white colony to verify. Extract adiponectin recombination vector named pCDNA3.1-ADPN.③The cultured L-02 cells are transfected with pCDNA3.1-ADPN by liposome, after sieving by G418. Immunofluorescence observes transfection efficiency. The expression product in cell lysate and culture supernatant was identified by SDS-PAGE、western blot and immunohistochemical method.④Using 0mmol/L、5 mmol/L、15mmol/L and 30mmol/L glucose to simulate no glucose,low glucose, middle glucose, high glucose environment. Treat transfect ion cells and control cells with the above-mentioned conditions. Determine cultivate glucose by enzymology; detect the recombination adiponectin protein's activity.⑤Determine phospho-AMPK, CREB, phospho-CREB in normal L-02 group, transfected group, high glucose treatment group, and hungry group by western blot, observed the TORC2 localization in the four group, glucose dehydrogenase couple way detectes Glucose-6-phosphorate (G6P) activity.
Results:①Successfully construct the adiponectin clone vector pMD18-T/ADPN and adiponectin eukaryotic expression vector pCDNA3.1/CT-GFP-TOPO-ADPN. ADPN gene was inserted in correct location.②We can see the green fluorescence in the transfected pCDNA3.1/CT-GFP- TOPO-AND cells by the immunofluorescence microscope; the transfection efficiency is about 60%.③Recombinant protein secretes into culture medium. The MW of recombinant protein is about 57kD. Western blot proves that expressed protein could combine with ADPN polyclonal antibody.④There is obvious glucose difference between transfection group and control group (p<0.05), which treated with high glucose for 24 h. There is no obvious among no glucose group, low glucose group and middle glucose group (p>0.05).⑤Compare with other groups, there is stronger p-AMPK expression in the transfect ion group (p<0.05), TORC2 was mainly localization in cytoplasm, the G6P activity was cut down significant (p<0.05), but there is no significant in the expression of CREB、p-CREB (p>0.05).
Conclusions:Adiponectin eukaryotic expression vector was successfully constructed. It can express solubility and secrete adiponectin protein in human hepatocytes L-02. The recombinant protein has biology activity.The mechanism of inhibitin hepar glyconeogenesis may involve activing AMPK, depressing the TORC2 entry the nuclear, and cutting down the G6P activity. The signaling pathway may be adiponectin-AMPK-CREB-TORC2- glyconeogenesis key enzyme.
引文
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2 Wullschleger S, Loewith R, Hall MN.TOR signaling in growth and metabolism. Cell,2006,124:471-484.
3 De Virgilio C,Loewith R.Cell growth control:little eukaryotes make big contribu-tions.Oncogene,2006,25:6392-6415.
4 Jacinto E,Loewith R,Schmidt A,et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive.Nat Cell Biol,2004,6:1122-1128.
5 Wullschleger S,Loewith R,Oppliger W,et al.Molecular organization of target of rapamycin complex 2.J Biol Chem,2005,280:30697-30704.
6 Sarbassov DD, Ali SM, Kim DH,et al.Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton.Curr Biol,2004,14:1296-1302.
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9 Canettieri G, Koo SH, Berdeaux R,et al.Dual role of the coactivator TORC2 in modulating hepatic glucose output and insulin signaling. CellMetab,2005,2: 331-338.
10 Ravnskjaer K, Kester H, Liu Y,et al. Cooperative interactions between CBP and TORC2 confer selectivity to CREB target gene expression.EMBO J,2007,26: 2880-2889.
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13 Sriwijitkamol A,Ivy JL,Christ-Roberts C,et al. LKB1-AMPK signaling in muscle from obese insulin-resistant Zucker rats and effects of training. Am J Physiol Endocrinol Metab,2006,290:925-932.
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1 Koo SH, Flechner L, Qi L, et al.The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism.Nature,2005,437(7062):1109-1111.
2 Wullschleger S, Loewith R, Hall MN.TOR signaling in growth and metabolism. Cell,2006,124:471-484.
3 De Virgilio C,Loewith R.Cell growth control:little eukaryotes make big contribu-tions.Oncogene,2006,25:6392-6415.
4 Jacinto E,Loewith R,Schmidt A,et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive.Nat Cell Biol,2004,6:1122-1128.
5 Wullschleger S,Loewith R,Oppliger W,et al.Molecular organization of target of rapamycin complex 2.J Biol Chem,2005,280:30697-30704.
6 Sarbassov DD, Ali SM, Kim DH,et al.Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton.Curr Biol,2004,14:1296-1302.
7 Reinke A, Anderson S, McCaffery JM, et al.TOR complex 1 includes a novel component, Tco89p (YPL180w), and cooperates with Ssdlp to maintain cellular integrity in Saccharomyces cerevisiae. J Biol Chem.,2004,279:14752-14762.
8 Montminy M, Koo SH, Zhang X. The CREB family:key regulators of hepatic metabolism. Ann Endocrinol,2004,65:73-75.
9 Canettieri G, Koo SH, Berdeaux R,et al.Dual role of the coactivator TORC2 in modulating hepatic glucose output and insulin signaling. CellMetab,2005,2: 331-338.
10 Ravnskjaer K, Kester H, Liu Y,et al. Cooperative interactions between CBP and TORC2 confer selectivity to CREB target gene expression.EMBO J,2007,26: 2880-2889.
11 Foretz M,Guigas B,Viollet B.From cancer to diabetes treatment:the tumor suppressor LKB1 as a new pharmacological target.Med Sci,2006,22:348-350.
12 Marx J. Cancer-suppressing enzyme adds a link to type 2 diabetes, Medicine, 2005,310:1259.
13 Sriwijitkamol A,Ivy JL,Christ-Roberts C,et al. LKB1-AMPK signaling in muscle from obese insulin-resistant Zucker rats and effects of training. Am J Physiol Endocrinol Metab,2006,290:925-932.
14 Sakamoto K, McCarthy A, Smith D,et al. Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction.EMBO J,2005, 24:1810-2020.
15 Shaw RJ, Lamia KA, Vasquez D,et al.The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin.Science,2005,310: 1642-1646.
16 Katoh Y, Takemori H, Min L,et al.Salt-inducible kinase-1 represses cAMP response element-binding protein activity both in the nucleus and in the cytoplasm.Eur J Biochem,2004,271:4307-19.
17 Al-Hakim AK, Goransson O, Deak M,et al.14-3-3 cooperates with LKB1 to regulate the activity and localization of QSK and SIK.J Cell Sci,2005,118: 5661-5673.